The present invention relates to a recombinant adenovirus comprising a chimeric adenoviral fiber protein and the use of a recombinant adenovirus comprising a chimeric adenoviral fiber protein in gene therapy.
Adenoviruses belong to the family Adenoviridae, which is divided into two genera, namely Mastadenovirus and Aviadenovirus. Adenoviruses are nonenveloped, regular icosahedrons 65-80 nm in diameter (Home et al., J. Mol. Biol., 1, 84-86 (1959)). The capsid is composed of 252 capsomeres of which 240 are hexons and 12 are pentons (Ginsberg et al., Virology, 28, 782-783 (1966)). The hexons and pentons are derived from three different viral polypeptides (Maizel et al., Virology, 36, 115-125 (1968); Weber et al, Virology, 76, 709-724 (1977)). The hexon comprises three identical polypeptides of 967 amino acids each, namely polypeptide II (Roberts et al., Science, 232, 1148-1151 (1986)). The penton contains a penton base, which is bound to the capsid, and a fiber, which is noncovalently bound to and projects from the penton base. The fiber protein comprises three identical polypeptides of 582 amino acids each, namely polypeptide IV. The adenovirus serotype 2 (Ad2) penton base protein is an 8xc3x979 nm ring-shaped complex composed of five identical protein subunits of 571 amino acids each, namely polypeptide III (Boudin et al., Virology, 92, 125-138 (1979)). Proteins IX, VI, and IIIa are also present in the adenoviral coat and are thought to stabilize the viral capsid (Stewart et al., Cell, 67, 145-154 (1991); Stewart et al., EMBO J., 12(7), 2589-2599 (1993)).
Once an adenovirus attaches to a cell, it undergoes receptor-mediated internalization into clathrin-coated endocytic vesicles of the cell (Svensson et al., J. Virol., 51, 687-694 (1984); Chardonnet et al., Virology, 40, 462-477 (1970)). Virions entering the cell undergo a stepwise disassembly in which many of the viral structural proteins are shed (Greber et al, Cell, 75, 477-486 (1993)). During the uncoating process, the viral particles cause disruption of the cell endosome by a pH-dependent mechanism (Fitzgerald et al., Cell, 32, 607-617 (1983)), which is still poorly understood. The viral particles are then transported to the nuclear pore complex of the cell (Dales et al., Virology, 56, 465-483 (1973)), where the viral genome enters the nucleus, thus initiating infection.
An adenovirus uses two separate cellular receptors, both of which must be present, to efficiently attach to and infect a cell (Wickham et al., Cell, 73, 309-319 (1993)). First, the Ad2 fiber protein attaches the virus to a cell by binding to an, as yet, unidentified receptor. Then, the penton base binds to xcex1v integrins, which are a family of a heterodimeric cell-surface receptors that mediate cellular adhesion to the extracellular matrix molecules fibronectin, vitronectin, laminin, and collagen, as well as other molecules (Hynes, Cell, 69, 11-25 (1992)), and play important roles in cell signaling processes, including calcium mobilization, protein phosphorylation, and cytoskeletal interactions (Hynes, supra).
The fiber protein is a trimer (Devaux et al., J. Molec. Biol., 215, 567-588 (1990)) consisting of a tail, a shaft, and a knob. The fiber shaft region is composed of repeating 15 amino acid motifs, which are believed to form two alternating b-strands and b-bends (Green et al., EMBO J., 2, 1357-1365 (1983)). The overall length of the fiber shaft region and the number of 15 amino-acid repeats differ between adenoviral serotypes. For example, the Ad2 fiber shaft is 37 nm long and contains 22 repeats, whereas the Ad3 fiber is 11 mn long and contains 6 repeats. The receptor binding domain of the fiber protein is localized in the knob region encoded by the last 200 amino acids of the protein (Henry et al., J. of Virology, 68(8), 5239-5246 (1994)). The regions necessary for trimerization are also located in the knob region of the protein (Henry et al. (1994), supra). A deletion mutant lacking the last 40 amino acids does not trimerize and also does not bind to penton base (Novelli et al. Virology, 185, 365-376 (1991)). Thus, trimerization of the fiber protein is necessary for penton base binding. Nuclear localization signals that direct the protein to the nucleus to form viral particles following its synthesis in the cytoplasm are located in the N-terminal region of the protein (Novelli et al. (1991), supra). The fiber, together with the hexon, are the main antigenic determinants of the virus and also determine the serotype specificity of the virus (Watson et al., J. Gen. Virol., 69, 525-535 (1988)). The fiber protein is glycosylated with single N-acetyl-glucosamine residues; however, the functional significance of the glycosylation remains unclear (Caillet-Boudin et al., Eur. J. Biochem., 184, 205-211 (1989)).
Over ten fiber proteins from different adenoviral serotypes have been sequenced, only to reveal a larger sequence diversity than that observed among other adenoviral proteins. For example, the knob regions of the fiber proteins from the closely related Ad2 and Ad5 serotypes are only 63% similar at the amino acid level (Chroboczek et al., Virology, 186, 280-285 (1992)), whereas their penton base sequences are 99% identical. Ad2 and Ad5 fiber proteins, however, both likely bind to the same cellular receptor, since they cross-block each other""s binding. In contrast, Ad2 and Ad3 fibers are only 20% identical (Signas et al., J. of Virology, 53, 672-678 (1985)) and presumably bind to different receptors, since each fails to cross-block the other""s binding (Defer et al., J. of Virology, 64(8), 3661-3673 (1990)). Ad3 fiber utilizes sialic acid as its receptor, whereas Ad2 fiber does not. Pretreatment of cells with neuraminidase or periodate abrogates Ad3, but not Ad2, binding. Also, soluble analogues of sialic acid block Ad3, but not Ad2, binding. However, sequence comparisons of the Ad2 and Ad3 fiber genes do show distinct regions of conservation. Most of these regions are also conserved in the other human adenoviral fiber genes. Nonhuman adenoviral fiber genes show less homology to human serotypes but still trimerize. The receptors used by nonhuman serotypes are unknown.
Recombinant adenoviral vectors have been used for the cell-targeted transfer of one or more recombinant genes to diseased cells or tissue in need of treatment. Such vectors are characterized by the advantage of not requiring host cell proliferation for expression of adenoviral proteins (Horwitz et al., In Virologv, Raven Press, New York, vol. 2, pp. 1679-1721 (1990); and Berkner, BioTechniques, 6, 616 (1988)), and, if the targeted tissue for somatic gene therapy is the lung, these vectors have the added advantage of being normally trophic for the respiratory epithelium (Straus, In Adenoviruses, Plenan Press, New York, pp. 451-496 (1984)).
Other advantages of adenoviruses as potential vectors for human gene therapy are as follows:
(i) recombination is rare; (ii) there are no known associations of human malignancies with adenoviral infections despite common human infection with adenoviruses; (iii) the adenoviral genome (which is a linear, double-stranded DNA) can be manipulated to accommodate foreign genes that range in size; (iv) an adenoviral vector does not insert its DNA into the chromosome of a cell, so its effect is impermanent and unlikely to interfere with the cell""s normal function; (v) the adenovirus can infect non-dividing or terminally differentiated cells, such as cells in the brain and lungs; and (vi) live adenovirus, having as an essential characteristic the ability to replicate, has been safely used as a human vaccine (Horwitz et al. (1990), supra; Berkner et al. (1988), supra; Straus et al. (1984), supra; Chanock et al., JAMA, 195, 151 (1966); Haj-Ahmad et al., J. Virol., 57, 267 (1986); and Ballay et al., EMBO, 4, 3861 (1985)).
A drawback to adenovirus-mediated gene therapy is that significant decreases in gene expression are observed after two weeks following administration of the vector. In many therapeutic applications the loss of expression requires re-administration of the viral vector to overcome losses in expression. However, following administration of the viral vector, neutralizing antibodies are raised against both the fiber and hexon proteins (Wohlfart, J. Virology, 62, 2321-2328 (1988); Wohlfart et al., J. Virology, 56, 896-903 (1985)). This antibody response against the virus then can prevent effective re-administration of the viral vector. Accordingly, recombinant adenoviral vectors capable of avoiding such neutralizing antibodies that would allow repeated doses of adenoviral vectors to be administered in the context of gene therapy would represent a significant advance in current gene therapy methodology.
Another drawback of using recombinant adenovirus in gene therapy is that all cells that express the aforementioned two receptors used by adenovirus to attach and infect a cell will internalize the gene(s) being administered not just the cells in need of therapeutic treatment. Likewise, certain cells, such as lymphocytes, which lack the xcex1v integrin adenoviral receptors, are impaired in the uptake of adenoviruses (Silver et al., Virology 165, 377-387 (1988); Horvath et al., J. of Virology, 62(1), 341-345 (1988)) and are not readily amenable to adenovirus-mediated gene delivery. Accordingly, limiting adenoviral entry to specific cells or tissues and/or expanding the repertoire of cells amenable to adenovirus-mediated gene therapy would be a significant improvement over the current technology. Targeted adenoviral gene delivery should expand the cells amenable to gene therapy, reduce the amount of adenoviral vector that is necessary to obtain gene expression in the targeted cells, and reduce side effects and complications associated with increasing doses of adenovirus, such as inflammation and the transfection of normal, healthy cells.
Attempts have been made to target a virus to specific cells by sterically blocking adenoviral fiber protein with antibodies and chemically linking tissue-specific antibodies to the viral particle (Cotten et al., Proc. Natl. Acad. Sci. USA, 89, 6094-6098 (1992)). Although this approach has demonstrated the potential of targeted gene delivery, the complexity and reproducibility of this approach present major hurdles blocking its application in clinical trials. The difficulties thus far encountered in targeting the virus by these methods involve the method of synthesis required, which is to make major alterations in the viral particles following their purification. These alterations involve additional steps that covalently link large molecules, such as polylysine, receptor ligands and antibodies, to the virus (Cotten (1 992), supra; Wagner et al., PNAS USA, 89, 6099-6103 (1992)). The targeted particle complexes are not homogeneous in structure and their efficiency is sensitive to the relative ratios of viral particles, linking molecules, and targeting molecules used.
The present invention seeks to overcome at least some of the aforesaid problems of recombinant adenoviral gene therapy. In one aspect, the present invention provides a recombinant adenoviral vectors capable of avoiding neutralizing antibodies upon repeat administration, thereby enabling the maintenance of recombinant gene expression at a therapeutically effective level. The present invention also relates to a cell-specific/tissue-specific recombinant adenovirus so as to target gene therapy to selected cells/tissues, thereby reducing the amount of recombinant adenoviral vector administered and any side-effects/complications. In another embodiment, the present invention provides means for modifying the viral particle at the level of gene expression, thus allowing viral particles to be purified by conventional techniques. In another embodiment, the present invention provides a method of gene therapy involving the use of such a homogeneous adenovirus, without the need for additional chemical modifications of viral particles, such as psoralen inactivation, or the addition of molecules to the virus which permit the covalent linkage of additional molecules to the virus. These and other objects and advantages of the present invention, as well as additional inventive features, will be apparent from the following detailed description.
The present invention provides a recombinant adenovirus comprising a chimeric fiber protein, which differs from the native (wild-type) fiber protein by the introduction of a nonnative amino acid sequence. The nonnative amino acid sequence allows the adenovirus to be targeted towards a protein, such as a receptor or a bi- or multi-specific protein, which is specific for binding to the nonnative amino acid sequence and a target receptor, by facilitating direct binding between the nonnative amino acid sequence and the protein, i.e., receptor or bi/multi-specific protein. Alternatively, the nonnative amino acid sequence facilitates proteolytic removal of the chimeric fiber protein to allow targeting of the adenovirus by means of another adenoviral coat protein, such as the penton base. The present invention also provides an adenoviral transfer vector, among others, comprising a recombinant fiber gene sequence for the generation of a chimeric fiber protein, and a method of using a protein-specific recombinant adenovirus, which is specific for a given receptor or bi-/multi-specific protein and which comprises a therapeutic gene, in gene therapy.